Optical sensors based on fluorescent or phosphorescent materials are known for use in detecting various gases and ionic materials in fluid samples, such as blood and sea water. Typically, oxygen sensors are based on quenching of the luminescence of luminescent materials within the sensors by the analyte gases in a sample. As described in MacCraith et al., J Sol-Gel Sci. and Tech., 8, pages 1053-1061 (1997) and references therein, the variation in the luminescence signal with analyte concentration is described by the Stern-Volmer equation: EQU I.sub.0 /I=1+K.sub.sv [analyte]
where I.sub.0 is the luminescence signal in the absence of the analyte and K.sub.sv is the Stern-Volmer quenching constant which determines sensitivity. For reliable analyses, it is preferable to achieve a linear relationship or response between the luminescence signal and the amount, such as partial pressure of a gas either in a gaseous or dissolved form.
Alternatively, such optical sensors may employ fluorescent dyes which change their absorbance properties and hence, indirectly alter their emission yield upon protonation or deprotonation according to the sample or internal sensor pH. Two examples of such sensors include a CO.sub.2 sensor with the dye hydroxypyrenetrisulfonic acid, as described in U.S. Pat. No. 5,506,148, and a pH sensor with the dye fluorescein, as described in WO 95-30148 to Alder, et al., both of which are incorporated by reference herein.
In these optical sensors, a substrate which is transparent to the excitation and emission wavelengths of the luminescent material is typically used. This type of substrate makes it possible to bring a thin sensor coating or layer containing the luminescent material on the substrate into contact with the sample while permitting the excitation light to reach the sensor coating and the emission signal generated by the luminescent material to be detected through the transparent substrate. In general, with this approach to optical sensors, it is sometimes difficult to achieve reliable analytical measurements because specific samples, such as blood and milk, tend to absorb, or scatter, or reflect the excitation light and the emission signal either back into the sensor detection layer or back into the detection optics through the transparent substrate. Other types of optical interference are stray light from the ambient conditions around the optical sensor and sample and stray light from a second optical sensor located in the vicinity of the first sensor as well as fluorescence from the bulk sample (i.e., bilirubin fluorescence in the case of blood).
One approach to solving this problem has been to place a second coating layer over the sensing or detection layer and thus interpose a separate layer between the sensing layer and the sample. This second coating layer typically absorbs and blocks the excitation light and emission signal to prevent them from reaching the sample. Thus, any sample-induced changes due to hematocrit effects on absorption, scattering, or reflection of the excitation light and emission signal are substantially reduced.
The use of a second layer over the sensing layer has been described for fluorescence based sensors utilizing chemical processes to produce an opaque but permeable second layer which is laminated or coated onto the sensing layer. For example, U.S. Pat. No. 5,091,800 to Offenbacher et al. discloses the use of ion permeable cover membranes made from crosslinked polyvinyl alcohol or cellophane which is stretched on a form and then impregnated with silver, gold, or platinum colloidal particles through a series of chemical treatments to form an opaque membrane. This opaque membrane is then laid in a separate process step over the sensing layer. U.S. Pat. Nos. 4,919,891; 5,075,127; 5,081,041; and 5,081,042 to Yafuso et al. describe other examples of opaque second layers over the sensing layer where the opaque second or cover layer is an ion permeable cover membrane impregnated with a non-reflective opaque material such as carbon black or, alternatively, is a coating of a cellulosic resin with non-reflective opaque materials such as copper phthalocyanine or carbon black.
These sensors do, however, have disadvantages related to requiring an additional layer in their production. The introduction of a second layer between the sample and the detection layer disadvantageously tends to block or inhibit the requisite contact between the analyte and the detection layer. To overcome this drawback, the second layer must be highly permeable to the analyte and adhere well to the detection layer. In addition to the increased expense of a second process step and permeability issues, the variations in material compositions and properties can make this second opaque layer especially problematical when considering material compatibilities between the sensing and opaque layers in production and between the opaque layer and the sample in use.
These extra complexities and disadvantages of a second or cover layer interposed between the sensing layer and the sample are multiplied in the event that two or more different sensing layers are present in a single optical sensor for the detection of two or more different analyzable materials or analytes, such as described in a copending U.S. patent application Ser. No. 09/009,917 entitled "Optical Sensor and Method of Operation," which is fully incorporated herein by reference and referred to hereinafter as the "Chiron Sensor Application," filed on even day herewith by the common assignee. Each of these different sensing layers would require a matched second or cover layer, probably different for each sensing layer. Also, the processing, chemistry, and permeability of the opaque second or cover layer adds complexity and would be more difficult to control on a consistent production basis.
An alternative type of optical sensor is based on differences in absorbance rather than differences in luminescence. These absorbance based sensors typically require the excitation light to be transmitted through the sensing layer after exposure of the layer to the sample being analyzed. The analysis is performed using changes in the transmission or absorbance spectrum of the sensor or, in the case of opaque samples, by using changes in the reflectance spectrum of the sensor. The detection process for absorbance based sensors is generally less complex than for luminescence sensors, for example, because only the absorbance wavelength enters into the analysis. There is no secondary emission wavelength so that complications due to light scattering effects are confined to a single wavelength. However, luminescence sensing is more specific and hence significantly more sensitive due in part to its dependence upon two separate wavelengths (i.e., the excitation and emission) rather than just one wavelength. It therefore operates at much lower analyte levels of detection and with smaller sample volumes than absorption analysis.
One variation of the absorbance based sensors is a multilayer design with a reagent layer where the analyte reacts with a reagent to form a detectable species which diffuses to a detection layer where its amount is measured, as described in U.S. Pat. No. 4,042,335 to Clement. It also contains additional layers, such as a spreading layer, for a total of 3 or more layers in the optical sensor. Typically, this type of optical sensor uses a reflectance based measurement system with a transmitting substrate in a slide frame holder. For the particular measurement requirements of these sensors, a separate light blocking layer containing a reflective opaque material, such as titanium dioxide (TiO.sub.2), as one of the multiple layers has been described in U.S. Pat. No. 3,992,158 to Przybylowicz et al. and in U.S. Pat. No. 4,042,335 to Clement; both of which are fully incorporated herein by reference; and in U.S. Pat. Nos. 4,781,890 and 4,895,704 and Eur. Pat. 142,849 B1, all to Arai et al. Alternatively, the opaque materials can be in the spreading layer, as described in U.S. Pat. No. 3,992,158 to Przybylowicz et al. U.S. Pat. No. 4,895,704 to Arai et al. describes the incorporation of light-scattering particulates, such as titanium dioxide, in a hydrophilic layer containing a reagent or containing a registration layer, to make the light transmittance in the range of 2.5 to 10% at the wavelengths used for measuring the detectable species.
It has been suggested by Klimant et al. in Anal Chem. 67, pages 3160-3166 (1995) that one might incorporate TiO.sub.2 into a luminescent sensing layer although they were unsuccessful in producing an oxygen sensor which had the required response speed with the opacity to efficiently block optical interference.